The generation of humoral and cell-mediated immunity is orchestrated by the interaction of activated helper T cells with antigen-presenting cells (“APCs”) and effector T cells. Activation of the helper T cells is not only dependent on the interaction of the antigen-specific T-cell receptor (“TCR”) with its cognate peptide-MHC ligand, but also requires the coordinate binding and activation by a number of cell adhesion and costimulatory molecules. See, e.g. Salazar-Fontana, L. I., and B. E. Bierer (2001) Curr. Opin. Hemat. 8:5.
One critical costimulatory molecule is CD154, a Type II transmembrane protein that is expressed on the surface of CD4+ T cells in an activation-dependent, temporally-restricted manner. CD154 is also expressed, following activation, on a subset of CD8+ T cells, basophils, mast cells, eosinophils, natural killer cells, B cells, macrophages, dendritic cells and platelets. The CD154 counter-receptor, CD40, is a Type I membrane protein that is constitutively and widely expressed on the surface of many cell types, including APCs. See, e.g., Foy, T. M. et al. (1996) Ann. Rev. Immunol. 14:591.
Signaling through CD40 by CD154 initiates a cascade of events that results in the activation of the CD40 receptor-bearing cells and optimal CD4+ T cell priming. More specifically, the binding of CD154 to CD40 promotes the differentiation of B cells into antibody secreting cells and memory B cells. See, e.g., Burkly, L. C. (2001) In Adv. Exp. Med. Bio., Vol. 489. D. M. Monroe et al. eds. Kluwer Academic/Plenum Publishers, p. 135 (hereafter “Burkly, supra”). Additionally, the CD154-CD40 interaction promotes cell-mediated immunity through the activation of macrophages and dendritic cells and the generation of natural killer cells and cytotoxic T lymphocytes. See, e.g., Burkly, ibid.
The pivotal role of CD154 in regulating the function of both the humoral and cell-mediated immune response has provoked great interest in the use of inhibitors of this pathway for therapeutic immunomodulation. As such, anti-CD154 antibodies have been shown to be beneficial in a wide variety of models of immune response to other therapeutic proteins or gene therapy, allergens, autoimmunity and transplantation. See, e.g., U.S. Pat. No. 5,474,771; Burkly, supra.
The CD40-CD154 interaction has been shown to be important in several experimentally induced autoimmune diseases where it has been shown that disease induction can be blocked with CD 154 antagonists at the time of antigen administration (Burkly, supra). The blockade of disease using anti-CD 154 antagonists has also been seen in animal models of spontaneous autoimmune disease. See, e.g., Burkly, supra.
There is currently a need for improved anti-CD154 antibodies with higher binding affinities and fewer unwanted side effects. Increased “effector functions” such as direct cytotoxicity, complement-dependent cytotoxicity (“CDC”), antibody-dependent cytotoxicity (“ADCC”) and abnormal antibody production, are unwanted side effects that may be associated with therapeutic antibodies.
Several antibody effector functions are mediated at least in part by Fc receptors (FcRs), which bind the Fc region of an antibody in the constant domain of a typical immunoglobulin. There are a number of Fc receptors which are specific for the different classes of immunoglobulins. The classes of immunoglobulins include IgG, IgE, IgA, IgM, and IgD. The classes of immunoglobulins are further divided into subclasses: IgG is divided into four subclasses (IgG1, IgG2, IgG3, and IgG4) and IgA is divided into two subclasses (IgA1 and IgA2). There are three known receptors for IgG: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16)). Each FcγR subclass is encoded by two or three genes, and alternative RNA splicing leads to multiple transcripts and a broad diversity in FcγR isoforms.
Typically, immunoglobulins are Y-shaped molecules comprising two identical heavy chains and two identical light chains. Disulfide bonds link together the heavy and light chain pairs as well as the two heavy chains. Each chain consists of one variable domain that varies in sequence and is responsible for antigen binding; these domains are known as the VH and VL domains for the heavy and light chains, respectively. In the light chain there is a single constant domain (CL) and in the heavy chain there are three constant domains (CH1, CH2 and CH3). Molecules containing all of the variable and constant domains may be referred to as whole antibodies.
The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter “Kabat et al., supra”). This numbering system is used in the present specification except where otherwise indicated. It should be noted that the Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence.
There are three regions within the variable domains that are hypervariable in sequence set within four more highly conserved framework regions. These hypervariable CDRs are primarily responsible for antigen recognition. The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Lesk, A. M. J. Mol. Biol., 1987, 196:901-917), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus ‘CDR-H1’, as used herein, also includes a CDR located at residues 26 to 35, as described by a combination of the Kabat numbering system and Chothia's topological loop definition. The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system.
Though naturally occurring antibodies, as whole antibodies or as fragments retaining specific binding properties, were originally derived from a single species, engineered antibodies may be derived from more than one species of animal, e.g., chimeric antibodies. To date, mouse (murine)/human chimeric and murine/non-human primate antibodies have principally been generated, though other hybrid species combinations are possible. Many different configurations of naturally occurring and engineered antibody polypeptides, and derivatives and fragments thereof, are now known. The feature common to all is that the polypeptide or polypeptides retain antigen-binding specificity through one or more epitope-binding domains. Aside from epitope binding, the functional properties of an antibody polypeptide may differ depending on what other sequences are present, e.g., Fc domains or other sequences that activate effector functions and/or interact with other cellular pathways.
CD154 binding proteins that comprise epitope-binding domains (such as CDRs or variable domains) incorporated into a non-immunoglobulin scaffold or framework (see, for example, Binz et al. 2005 Nat Biotech 23: 1257-1268; Hosse et al. 2006 Protein Science 15: 14-27) may exhibit reduced effector functions. It would be desirable to have new binding proteins that specifically antagonize CD154 binding to CD40 and reduce or eliminate downstream functions of the CD154-CD40 complex. It would also be desirable to have CD154 binding proteins, such as anti-CD154 antibodies, with reduced effector functions compared to known anti-CD154 antibodies.